(a). Field of the Invention
The present invention relates to a device and a method for RNA separation, more particularly, to a device and a method capable of rapidly purifying mRNA for prokaryotic cells.
(b). Description of the Prior Arts
Up to today, biotechnology development is already having a significant advance on biochemical analysis. It is clear that the knowledge of those molecular biology techniques provide a powerful resource for studying biological processes, and it is also cleaning the way for future research. In particular, we can know more detail about different pathogens, such as facts about bacteria and virus, development and process of disease variation, even the amplification and exhibition of human cells, basing on gene modulation and exhibition.
In order to efficiently study the gene expression, a way must be developed to purify the mRNA of target gene of cell or pathogenesis. After then, we can advance to know the physiological and biochemical roles of target genes according to the quantity, the length of half life and the mechanism of biochemical interaction. There are three kinds of ribonucleic acid, such as the message ribonucleic acid, ribosome ribonucleic acid and transfer ribonucleic acid in the prokaryotic and eukaryotic cells. But the amount of mRNA is much less than the tRNA and rRNA. Therefore, a special biotechnology is needed to isolate the mRNA.
Most of the previous methods for isolating mRNA can be divided into two steps, i.e. the total RNA extraction and the mRNA purification. The mechanism of total RNA extraction is based on that deoxyribonucleic acid will denature at lower pH 7 and protein will also denature in the phenol, therefore, the DNA and protein will denature and combine together in the acid phenol solution. The traditional protocol of preparation of total RNA is to pellet up to 107 cells in a suitable tube by centrifugation at 400 g for 5 min at 4° C. After the supernatant is being poured off, the pellet is disrupted by flicking the base of the tube and therefore the cells are re-suspended in the Trizol reagent. Afterward, the cells are transferred to a microcentrifuge tube and incubated in the Trizol for 5 min at room temperature, further, the cells are stirred occasionally to ensure that cells are completely disrupted. Following, to each 0.5 ml of cells in Trizol, adding 10 ul of chloroform, mixing well by shaking the solution for 15 second and leaving at room temperature for 2-3 min, then the solution is centrifuge at 14,500 g for 15 min at 4° C. In order to take the upper aqueous phase that contains the RNA to a new tube, first, 250 ul isopropanol is added to the centrifuged solution and keeps thereof at 4° C. following by pouring off the supernatant, re-spinning for 1 min and removing the residual supernatant. Secondly, the pellet is washed with 1 ml 75% ethanol, and is stirred and centrifuged at 9,000 g for 5 min at 4° C. Finally, removing the ethanol, allowing the pellet to air dry, and re-suspending the pellet in 75 ul of double-deionized, RNase-free water.
The total RNA solution is poured into the affinity chromatography column which contains the specific oligo (dt) cellulose (as seen in FIG. 1). The column is then washed twice in modulated volume of wash buffer, thus, the tRNA, rRNA and other residual will be eluted. Finally, eluting the mRNA from the column with modulate elution buffer, then the pure mRNA is isolated. But there are disadvantages in the aforesaid protocols of mRNA purification.    (1) The range of application is narrow. That is, the traditional design is suitable to the eukaryotic cells, but not suit for the prokaryotic cells. Because the mRNA of prokaryotic cells without the pol (A) tail structure, mRNA can not to be separated between tRNA and rRNA by the hydrogen-bond binding of Adenine and Thymidine in the affinity column.    (2) The protocols are complicated, costly and time consuming to operate. Beside, it is easy to suffer the contamination of RNase during the operation.    (3) The RNA separation apparatus takes up a lot of laboratory space. Furthermore, the isolated mRNA sample can not be tested continuously in the biochemical analysis and automation process.     As the above description, the traditional protocols of mRNA purification still exist some space of improvements.